Mitogen-activated protein kinase MEK6 and methods of use therefor

ABSTRACT

Compositions and methods are provided for potentiating the activity of the mitogen-activated protein kinase p38. In particular the mitogen-activated protein kinase kinase MEK6, and variants thereof that stimulate phosphorylation of p38 are provided. Such compounds may be used, for example, for therapy of diseases associated with the p38 cascade and to identify antibodies and other agents that inhibit or activate signal transduction via p38.

TECHNICAL FIELD

[0001] The present invention relates generally to compositions andmethods for modulating the activity of the mitogen-activated proteinkinases, including p38. The invention is more particularly related tothe mitogen-activated protein kinase kinase MEK6 and variants thereofthat stimulate phosphorylation and activation of substrates, such asp38, and to the use of compounds, for example, to activate p38 and toidentify antibodies and other agents that inhibit or activate signaltransduction via the p38 kinase cascade.

BACKGROUND OF THE INVENTION

[0002] Mitogen-activated protein kinases (MAPKs) are members ofconserved signal transduction pathways that activate transcriptionfactors, translation factors and other target molecules in response to avariety of extracellular signals. MAPKs are activated by phosphorylationat a dual phosphorylation motif with the sequence Thr-X-Tyr bymitogen-activated protein kinase kinases (MAPKKs). In higher eukaryotes,the physiological role of MAPK signaling has been correlated withcellular events such as proliferation, oncogenesis, development anddifferentiation. Accordingly, the ability to regulate signaltransduction via these pathways could lead to the development oftreatments and preventive therapies for human diseases associated withMAPK signaling, such as inflammatory diseases, autoimmune diseases andcancer.

[0003] In mammalian cells, three parallel MAPK pathways have beendescribed. The best characterized pathway leads to the activation of theextracellular-signal-regulated kinase (ERK). Less well understood arethe signal transduction pathways leading to the activation of the cJunN-terminal kinase (JNK) and the p38 MAPK (for reviews, see Davis, TrendsBiochem. Sci. 19:470473 (1994); Cano and Mahadevan, Trends Biochem. Sci.20:117-122(1995)). The identification and characterization of members ofthese cascades is critical for understanding the signal transductionpathways involved and for developing methods for activating orinactivating MAPKs in vivo.

[0004] Two MAPKKs capable of activating p32 in vitro have been described(see Derijard et al., Science 267:682-685 (1995)). MKK3 appears to bespecific for p38(i.e., does not activate JNK or ERK), while MKK4activates both p38 and JNK. MKK3 and MKK4 also stimulate thephosphorylation of p38 in certain cell lines after treatment withstimuli, such as ultraviolet radiation and NaCl. However, a stronger andmore specific in vivo stimulator of p38 phosphorylation would havegreater utility in therapeutic methods.

[0005] Accordingly, there is a need in the art for improved methods formodulating p38 activity and related enzymes or kinases in vivo, and fortreating diseases associated with the p38 signal transduction pathway.The present invention fulfills these needs and further provides otherrelated advantages.

SUMMARY OF THE INVENTION

[0006] Briefly stated, the present invention provides compositions andmethods for modulating the activity of the mitogen-activated proteinkinase (IAPK) p38. In one aspect, the present invention providespolypeptides comprising the amino acid sequence provided in SEQ ID NO:2or a variant thereof that differs only in conservative substitutionsand/or modifications at no more than 10% of the amino acid residues Suchvariants include constitutively active polypeptides. In a relatedaspect, polypeptides comprising the amino acid sequence provided in SEQID NO:2 modified at no more than 10% of the amino acid residues, suchthat the polypeptides are rendered constitutively inactive, areprovided.

[0007] In other aspects, isolated DNA molecules encoding polypeptides asdescribed above, as well as recombinant expression vectors comprisingsuch DNA molecules and host cells transformed or transfected with suchexpression vectors, are provided.

[0008] In further aspects, the present invention provides methods forphosphorylating p38, comprising contacting p38 with a polypeptide asdescribed above, and for activating a member of the p38 cascade in anorganism, comprising admimistering to an organism a polypeptide asdescribed above. In a related aspect, the present invention providesmethods for treating a patient afflicted with a disease associated withthe p38 cascade, comprising administering to a patient a compound thatpromotes or inhibits the phosphorylation of p38 by MEK6.

[0009] Methods are also provided for screening for agents that inhibitor stimulate signal transduction via the p³⁸ cascade. Such methodscomprise: (a) contacting a candidate acen. with a polypeptide asdescribed above; and (b) subsequently measuring the ability of thepolypeptide to activate p38. In yet another aspect, monoclonalantibodies that bind to a polypeptide as described above are providedWithin further aspects, the present invention provides methods and kitsfor detecting MEK6 kinase activity in a sample. The methods compriseevaluating the ability of the sample to phosphorylate p38, therebydetecting MEK6 kinase activity in the sample. The kits for detectingMEK6 kinase activity in a sample comprise p38 in combination with asuitable buffer.

[0010] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1. presents the nucleotide and primary amino acid sequence ofMEK6, as deduced from the sequence of cDNA clones isolated from a humanMOLT4 cDNA library. For the amino acid sequence, standard one-lettercodes are utilized.

[0012]FIGS. 2A and 2B are autoradiograms that depict Northern blotanalyses of the expression of human MKK3 (FIG. 2A) and human MEK6 (FIG.2B) mRNA in selected adult human tissues. The position of RNA sizemarkers in kb is shown on the left.

[0013]FIGS. 3A and 3B are autoradiograms that present the results ofkinase assays evaluating the substrate specificity of MEK6. FIG. 3Ashows the level of autophosphorylation of the substrates GST (lane 1),GST-JNK2 (lane 2), GST-p38 (lane 3) and His-ERK1 [K52R] (lane 4) and thelevel of phosphorylation by GST-MEK6 of the substrates GST (lane 5),GST-JNK2 (lane 6), GST-p38 (lane 7) and His-ERK1 [K52R] (lane 8). FIG.3B shows the results of a coupled kinase assay in which purified GST orGST-MEK6 was incubated with purified GST-MEK2, GST-p38 or GST in thepresence of ATP. The proteins were isolated and washed, and thenincubated with GST-cJun(1-79) (lanes 1-3) or GST-ATF2 (lanes 4-6) in thepresence of [γ-³²P]ATP. Reactions were separated by SDS-PAGE andvisualized by autoradiography. The position of protein molecular weightmarkers in kDa is shown on the left.

[0014]FIG. 4 is a graph depicting the relative levels of MEK6 kinaseactivity in HeLa cells transiently transfected with epitope-tagged MEK6and treated with anisomycin (50 ng/ml) or UV (254 nm; 120 J/ml) for thetimes indicated. The relative level of MEK6 activity in untreated cellswas arbitrarily assigned to be 1.

[0015]FIG. 5 is an autoradiogram and graph presenting the relativelevels of MEK6 kinase activity in HeLa cells transiently transfectedwith epitope-tagged MEK6 and activated for 40 min with 20 to 120 J/m² UV(254 nm) as indicated. Reactions were separated by SDS-PAGE andvisualized by autoradiography. The position of protein molecular weightmarkers in kDa is shown on the left. MEK6 activity was quantitated witha phosphorimager and ImageQuant software and is shown in the bar graph.

[0016]FIG. 6 is an autoradiogram depicting the relative levels of MEK6kinase activity in HeLa cells transiently transfected withepitope-tagged MEK6 (lanes 1-8) or the empty expression vector SRα3(lanes 9-16) and treated for 45 min with Anisomycin (An., 50 ng/ml) orleft untreated (ctrl) as indicated. The position of protein molecularweight markers in kDa is illustrated on the left The position of p38,ATF2 and an unknown protein (*) is indicated on the right.

[0017]FIGS. 7A and 7B are autoradiograms and graphs showing the relativelevels of MEK6 kinase activity in HeLa cells (FIG. 7A) or COS cells(FIG. 7B) transiently transfected with epitope-tagged MEK6 (lanes 1 to12) or the empty expression vector SRα3 (lanes 13 to 16) and treated for45 min with IL-1β (10 ng/ml), TNF-α (10 ng/mM), EGF (50 ng/ml), NGF (50ng/ml), PMA (50 ng/ml), Anisomycin (50 ng/ml), Cycloheximide (CX, 50ng/ml), Arsenite (200 μM), NaCl (200 μM) or UV (254 nm; 120 J/m²) orcotransfected with 1000 ng CMV5-MEKK as indicated. The position ofprotein molecular weight markers in kDa is illustrated on the left. MEK6activity depicted in the graphs was quantitated with a phosphorimagerand ImageQuant software.

[0018]FIG. 8 is an autoradiogram showing the relative levels of MEK6kinase activity in COS cells transiently transfected with epitope-taggedMEK6 (lanes 1 to 7) or JNKK (lanes 8 to 12) and increasing amounts ofCMV5-MEKK expression vector as indicated. The position of proteinmolecular weight markers in kDa is illustrated on the left. The positionof p38 and JNK2 is indicated on the right.

DETAILED DESCRIPTION OF THE INVENTION

[0019] As noted above, the present invention is generally directed tocompositions and methods for modulating (i.e., stimulating orinhibiting) the activity of the mitogen-activated protein kinase (MAPK)p38. Compositions that activate p38 generally stimulate p38phosphorylation. Such compositions include polypeptides comprising thehuman mitogen-activated protein kinase kinase (MAPKK) MEK6, or a variantthereof that retains the ability to stimulate p38 phosphorylation.Alternatively, compositions that activate p38 may include nucleic acidsequences that encode MEK6 or a variant thereof. Polypeptide variantswithin the scope of the present invention differ from MEK6 in one ormore conservative substitutions and/or modifications, at no more than10% of the amino acid residues in the native polypeptide, such that theability of the variant to stimulate p38 phosphorylation is notsubstantially diminished. Conservative substitutions may be made innoncritical and/or critical regions of the native protein. Variants mayalso, or alternatively, contain other conservative modifications,including the deletion or addition of amino acids that have minimalinfluence on the activity of the polypeptide. In particular, variantsmay contain additional amino acid sequences at the amino and/or carboxytermini. Such sequences may be used, for example, to facilitatepurification or detection of the polypeptide.

[0020] Compositions that stimulate p38 phosphorylation (therebyactivating p38) may also, or alternatively, include one or more agentsthat stimulate MEK6 kinase activity. Such agents include, but are notlimited to, stress-inducing signals (e.g, UV, osmotic shock,DNA-damaging agents), anisomycin, LPS, and cytokines, and may beidentified by combining a test compound with MEK6 in vitro andevaluating the effect of the test compound on the MEK6 kinase activityusing, for example, a representative assay described herein.

[0021] Compositions that inactivate p38 generally inhibit p38phosphorylation. Such compositions may include one or more agents thatinhibit or block MEK6 activity, such as an antibody that neutralizesMEK6, a competing peptide that represents the substrate binding domainof MEK6 or the dual phosphorylation motif of the MEK6 substrate, anantisense polynucleotide or ribozyme that interferes with transcriptionand/or translation of MEK6, a molecule that inactivates MEK6 by bindingto the kinase, a molecule that binds to the MEK6 substrate and preventsphosphorylation by MEK6 or a molecule that prevents transfer ofphosphate groups from the kinase to the substrate. Alternatively, anagent that inactivates p38 may inhibit the kinase activity ofphosphorylated p38.

[0022] Agents that inhibit MEK6 kinase activity may be identified bycombining a test compound with MEK6 in vitro and evaluating the activityof the MEK6 using a MEK6 kinase assay. Agents that inhibit the activityof phosphorylated p38 may similarly be identified by combining a testcompound with phosphorylated p38 and evaluating the effect of the testcompound on the p38 kinase activity using, for example, one of therepresentative assays described herein.

[0023] DNA sequences encoding native NLEK6 may be prepared byamplification from a suitable human cDNA library, using, polymerasechain reaction (PCR) and methods well known to those of ordinary skillin the art. For example, an adapter-ligated cDNA library prepared fromunstimulated Jurkat T cells may be screened using the 5′ specificforward primer 5′-TTGTGCTCCCCTCCCCCATCAAA GGAA-3′ and anadapter-specific primer. The resulting 1.6 kb cDNA has the sequenceprovided in SEQ ID NO:1. The encoded MEK6 polypeptide, shows in SEQ IDNO:2, has a predicted size of 334 amino acids, with a calculatedmolecular weight of 37.5 kD. MEK6 is 88% identical to its closesthomolog MKK3, and all relevant kinase subdomains are conserved. As shownin FIG. 1, the most divergent regions are the N-terminal region, with anadditional 18 amino acids, and the C-terminal region.

[0024] Polypeptides of the present invention may be prepared byexpression of recombinant DNA encoding the polypeptide in cultured hostcells. Preferably, the host cells are bacteria, yeast,baculovirus-infected insect cells or mammalian cells. The recombinantDNA may be cloned into any expression vector suitable for use within thehost cell, using techniques well known to those of ordinary skill in theart.

[0025] The DNA sequences expressed in this manner may encode MEK6, ormay encode portions or other variants of MEK6. DNA molecules encodingvariants of MEK6 may generally be prepared using standard mutagenesistechniques, such as oligonucleotide-directed site-specific mutagenesis,and sections of the DNA sequence may be removed to permit preparation oftruncated polypeptides. As noted above, up to 10% of the amino acidresidues may contain substitutions or other modifications, and any suchchanges preferably should not diminish the ability of the variant tostimulate p38 phosphorylation. In general, modifications may be morereadily made in non-critical regions, which are regions of the nativesequence that do not change the properties of MEK6. Non-critical regionsmay be identified by modifying the MEK6 sequence in a particular regionand assaying the ability of the resulting variant in a kinase assay,using p38 as a substrate, as described herein.

[0026] As noted above, MEK6 may also be modified by the addition ofsequences at the N- and/or C-terminus. For example, epitopes such as GST(glutathione-S-transferase), HA (hemagglutinin)-tag, FLAG andHistidine-tag may be added using techniques well known to those ofordinary skill in the art.

[0027] Modifications may also be made in critical regions of MEK6,provided that the resulting variant retains the ability to stimulate p38phosphorylation. Critical regions include the ATP binding site Lys⁶⁹,and the dual phosphorylation motif (Ser²⁰⁷, Thr²¹¹). The effect of anymodification on the ability of the variant to stimulate p38phosphorylation may generally be evaluated using any assay for MEK6kinase activity, such as the representative assays described herein.

[0028] Variants of MEK6 include constitutively active proteins. Ingeneral, activation of MEK6 in vivo requires stimulation by cvtokines,LTV, stress-inducing agents or osmotic shock. Constitutively activevariants display the ability to stimulate p³⁸ phosphorylation in theabsence of such stimulation. Such variants may be identified using therepresentative in vivo assays for MEK6 kinase activity described herein.Preferred constitutively active variants include polypeptides in whichthe phospho-acceptor amino acids within the MEK6 dual phosphorylationmotif (Ser²⁰⁷ and Thr²¹¹) are replaced with negatively charged aminoacids such as glutataic acid or aspartic acid.

[0029] MEK6 may also be modified so as to render the proteinconstitutively inactive (ie., unable to phosphorylate p38 even whenstimulated as described above). For example, mutation of the conservedlysine in kinase subdomain I has been found to render MAPKKs inactive.Accordingly, a preferred constitutively inactive variant contains amodification of Lys⁶⁹ in kinase subdomain I of MEK6. Other suchmodifications may be identified using the representative assaysdescribed herein. Genes encoding proteins modified so as to beconstitutively active or inactive may generally be used in replacementtherapy for treatment of a variety of disorders, as discussed in moredetail below.

[0030] Expressed polypeptides of this invention are generally isolatedin substantially pure form. Preferably, the polypeptides are isolated toa purity of at least 80% by weight, more preferably to a purity of atleast 95% by weight, and most preferably to a purity of at least 99% byweight. In general, such purification may be achieved using, forexample, the standard techniques of ammonium sulfate fractionation,SDS-PAGE electrophoresis, and affinity chromatography.

[0031] The present invention also provides methods for detecting thelevel of MEK6 in a sample, as well as for detecting MEK6 kinase activityin a sample. The level of MEK6, or nucleic acid encoding MEK6, maygenerally be determined using a reagent that binds to the MEK6 protein,DNA or RNA. To detect nucleic acid encoding MEK6, standard hybridizationand/or PCR techniques may be employed using a nucleic acid probe or aPCR primer. Suitable probes and primers may be designed by those ofordinary skill in the art based on the MEK6 cDNA sequence provided inSEQ ID NO: 1. To detect MEK6 protein, the reagent is typically anantibody, which may be prepared as described below. There are a varietyof assay formats known to those of ordinary skill in the art for usingan antibody to detect a polypeptide in a sample. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. For example, the antibody may be immobilized on a solid supportsuch that it can bind to and remove the polypeptide from the sample. Thebound polypeptide may then be detected using a second antibody thatbinds to the antibody/peptide complex and contains a detectable reportergroup. Alternatively, a competitive assay may be utilized, in whichpolypeptide that binds to the immobilized antibody is labeled with areporter group and allowed to bind to the immobilized antibody afterincubation of the antibody with the sample. The extent to whichcomponents of the sample inhibit the binding of the labeled polypeptideto the antibody is indicative of the level of polypeptide within thesample. Suitable reporter groups for use in these methods include, butare not limited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin.

[0032] MEK6 kinase assays, for use in evaluating the polypeptidevariants and other agents discussed above, include any assays thatevaluate a compound's ability to phosphorylate p38, thereby renderingthe p38 active (ie., capable of phosphorylating in vivo substrates suchas ATF2). p38 for use in such methods may be endogenous, purified orrecombinant, and may be prepared using any of a variety of techniquesthat will be apparent to those of ordinary skill in the art. Forexample, cDNA encoding p38 may be cloned by PCR amplification from asuitable human CDNA library, using primers based on the publishedsequence (Han et al., Science 265:808-811 (1994); Lee et al., Nature372:739-746 (1994)). p38 CDNA may then be cloned into a bacterialexpression vector and the protein produced in bacteria, such as E. coli,using standard techniques. The bacterial expression vector may, but neednot, include DNA encoding an epitope such as glutathione-S trasferaseprotein (GST) such that the recombinant protein contains the epitope atthe N- or C-terminus.

[0033] A MEK6 kinase assay may be performed substantially as describedin Derijard et al., Cell 76:1025-1037 (1994) and Lin et al., Science268:286-290 (1995), with minor modifications. Briefly, a polypeptidevariant of MEK6 may be incubated with p38 and [γ-³²P]ATP in a suitablebuffer (such as 20 mM HEPES (pH 7.6), 5 mM MnCl₂, 10 mM MgCl₂, 1 mMdithiothreitol) for 30 minutes at 30° C. In general, approximately 0.5μg of the variant and 1 μg recombinant p38, with 50 nM [γ-³²P]ATP, issufficient. Proteins may then be separated by SDS-PAGE on 10% gels andsubjected to autoradiography. Incorporation of [³²P]phosphate into p38may be quantitated using techniques well known to those of ordinaryskill in the art, such as with a phosphorimager. To evaluate thesubstrate specificity of polypeptide variants, a kinase assay maygenerally be performed as described above except that other MAPKsubstrates (i.e., JNK2 and/or ERK) are substituted for the p38.

[0034] To determine whether p38 phosphorylation results in activation, acoupled in vitro kinase assay may be performed using a substrate forp38, such as ATF2, with or without an epitope tag. ATF2 for use in suchan assay may be prepared as described in Gupta et al., Science267:389-393 (1995). Briefly, following phosphorylation of p38 asdescribed above, isolation of the protein by binding to GSH-sepharoseand washing with 20 mM HEPES (pH 7.6), 20 mM MgCl₂, the p38 (0.1-10 μg)may be incubated with ATF2 (0.1-10 μg) and [γ-³²P]ATP (10-500 nM) in abuffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl₂. It should be notedthat alternative buffer may be used and that buffer composition can varywithout significant effects on kinase activity. Reactions may beseparated by SDS-PAGE, visualized by autoradiography and quantitatedusing any of a variety of known techniques. Activated p38 will becapable of phosphorylating ATF2 at a level of at least 5% abovebackground using this assay.

[0035] To evaluate the effect of an antibody or other candidatemodulating agent on MEK6 activity, a kinase assay may be performed asdescribed above, except that MEK6 (rather than a variant thereof isgenerally employed and the candidate modulating agent is added to theincubation mixture. The candidate agent may be preincubated with MEK6kinase before addition of ATP and substrate. Alternatively, thesubstrate may be preincubated with the candidate agent before theaddition of kinase. Further variations include adding the candidateagent to a mixture of kinase and ATP before the addition of substrate,or a mixture of substrate and ATP before the addition of MEK6 kinase,respectively. All these assays can further be modified by removing thecandidate agent after the initial preincubation step. In general, asuitable amount of antibody or other candidate agent for use in such anassay ranges from about 0.1 μM to about 10 μM. The effect of the agenton MEK6 kinase activity may then be evaluated by quantitating theincorporation of [³²P]phosphate into p38, as described above, andcomparing the level of incorporation with that achieved using MEK6without the addition of the candidate agent.

[0036] MEK6 activity may also be measured in whole cells transfectedwith a reporter gene whose expression is dependent upon the activationof ATF2. For example, cells may be transfected with an ATF2-dependentpromoter linked to a reporter gene such as luciferase. In such a system,expression of the luciferase gene (which may be readily detected usingmethods well known to those of ordinary skill in the art) depends uponactivation of ATF2 by p38, which may be achieved by the stimulation ofMEK6 with an activator or by cotransfection with an expression vectorthat produces a constitutively active variant of MEK6. Candidatemodulating agents may be added to the system, as described below, toevaluate their effect on MEK6 activity.

[0037] Alternatively, a whole cell system may employ only thetransactivation domain of ATF2 fused to a suitable DNA binding domain,such as GHF-1 or GAL4. The reporter system may then comprise theGH-luciferase or GAL4-luciferase plasmid. Candidate MEK6 modulatingagents may then be added to the system to evaluate their effect onATF2-specific gene activation

[0038] In other aspects of the subject invention, methods for using theabove polypeptides to phosphorylate and activate p38, or peptidederivatives thereof, are provided. p38 substrate for use in such methodsmay be prepared as described above. In one embodiment, p38 may bephosphorylated in vitro by incubating p38 with MEK6, or a variantthereof, and ATP in a suitable buffer as described above for 30 minutesat 30° C. In general, the amounts of the reaction components may rangefrom about 0.1 μg to about 10 μg of MEK6 or a variant thereof, fromabout 0.1 μg to about 10 μg of recombinant p38, and from about 10 nM toabout 500 nM of ATP. Phosphorylated proteins may then be purified bybinding to GSH-sepharose and washing. The extent of p38 phosphorylationmay generally be monitored by adding [γ-³²P]ATP to a test aliquot, andevaluating the level of p38 phosphorylation as described above. Theactivity of the phosphorylated p38 may be evaluated using a coupled invitro kinase assay, as described above.

[0039] Once activated in vitro, p38 may be used, for example, toidentify agents that inhibit the kinase activity of p38. Such inhibitoryagents, which may be antibodies or drugs, may be identified using thecoupled assay described above. Briefly, a candidate agent may beincluded in the mixture of p38 and ATF2, with or without pre-incubationwith one or more components of the mixture, as described above. Ingeneral, a suitable amount of antibody or other agent for use in such anassay ranges from about 0.1 μM to about 10 μM. The effect of the agenton p38 kinase activity may then be evaluated by quantitating theincorporation of [³²P]phosphate into ATF2, as described above, andcomparing the level of incorporation with that achieved using activatedp38 without the addition of a candidate agent.

[0040] The above polypeptides and/or modulating agents may also be usedto phosphorylate, and thereby activate. p38 in a patient. As usedherein, a “patient” may be any mammal, including a human, and may beafflicted with a disease associated with the p38 cascade or may be freeof detectable disease. Accordingly, the treatment may be of an existingdisease or may be prophylactic. Diseases associated with the p38 cascadeinclude any disorder which is etiologically linked to MEKK6 kinaseactivity, including immune-related diseases (e.g. inflammatory diseases,autoimnnune diseases, malignant cytokine production or endotoxic shock),cell growth-related diseases (e.g., cancer, metabolic diseases, abnormalcell growth and proliferation or cell cycle abnormalities) and cellregeneration-related diseases (e.g., cancer, degenerative diseases,trauma, environmental stress by heat, UV or chemicals or abnormalitiesin development and differentiation).

[0041] For administration to a patient, one or more polypeptides and/ormodulating agents are generally formulated as a pharmaceuticalcomposition, formulated as a sterile aqueous or non-aqueous solution,suspension or emulsion, which additionally comprises a physiologicallyacceptable carrier (ie., a non-toxic material that does not interferewith the activity of the active ingredient). Any suitable carrier knownto those of ordinary skill in the art may be employed in thepharmaceutical compositions of this invention. Representative carriersinclude physiological saline solutions, gelatin, water, alcohols,natural or synthetic oils, saccharide solutions, glycols, injectableorganic esters such as ethyl oleate or a combination of such materials.Optionally, a pharmaceutical composition may additionally containpreservatives and/or other additives such as, for example, antimicrobialagents, anti-oxidants, chelating agents and/or inert gases.

[0042] Alternatively, a pharmaceutical composition may contain DNAencoding a polypeptide as described above, such that MEK6 or a variantthereof is generated in situ, in combination with a physiologicallyacceptable carrier. In such pharmaceutical compositions, the DNA may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid, bacterial and viralexpression systems, as well as colloidal dispersion systems, includingliposomes. Appropriate nucleic acid expression systems contain thenecessary DNA sequences for expression in the patient (such as asuitable promoter and terminating signal). The DNA may also be “naked,”as described, for example, in Ulmer et al., Science 259:1745-1749(1993).

[0043] Various viral vectors that can be used to introduce a nucleicacid sequence into the targeted patient's cells include, but are notlimited to, vaccine or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Preferably, the retroviralvector is a derivative of a murine or avian retrovirus including, butnot limited to, Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor-virus (MuMTV), and RousSarcoma Virus (RSV). A retroviral vector may additionally transfer orincorporate a gene for a selectable marker (to aid in the identificationor selection of transduced cells) and/or a gene that encodes the ligandfor a receptor on a specific target cell (to render the vector targetspecific). For example, retroviral vectors can be made target specificby inserting a nucleotide sequence encoding a sugar, a glycolipid, or aprotein. Targeting may also be accomplished using an antibody, bymethods known to those of ordinary skill in the art.

[0044] Viral vectors are typically non-pathogenic (defective),replication competent viruses, which require assistance in order toproduce infectious vector particles. This assistance can be provided,for example, by using helper cell lines that contain plasmids thatencode all of the structural genes of the retrovirus under the controlof regulators sequences within the LTR, but that are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Such helper cell lines include (butare not limited to) Ψ2. PA317 and PA12. A retroviral vector introducedinto such cells can be packaged and vector virion produced. The vectorvirions produced by this method can then be used to infect a tissue cellline, such as NMH 3T3 cells, to produce large quantities of chimericretroviral virons.

[0045] Another targeted delivery system for MEK6 polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. A preferred colloidal system for use as adelivery vehicle in vitro and in vivo is a liposome (i.e., an artificialmembrane vesicle). It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial sells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present. (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques 6:882, 1988).

[0046] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0047] Routes and frequency of administration and polypeptide,modulating agent or nucleic acid doses will vary from patient topatient. In general, the pharmaceutical compositions may be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity or transdermally. Between 1 and 6 doses may be administereddaily. A suitable dose is an amount of polypeptide or DNA that issufficient to show improvement in the symptoms of a patient afflictedwith a disease associated with the p38 cascade. Such improvement may bedetected based on a determination of relevant cytokine levels (e.g.,IL-2, IL-8), by monitoring inflammatory responses (e.g., edema,transplant rejection, hypersensitivity) or through an improvement inclinical symptoms associated with the disease. In general, the amount ofpolypeptide present in a dose, or produced in situ by DNA present in adose, ranges from about 1 μg to about 250 μg per kg of host, typicallyfrom about 1 μg to about 60 μg. Suitable dose sizes will vary with thesize of the patient, but will typically range from about 10 mL to about500 mL for 10-60 kg animal.

[0048] The MEK6 protein kinase described herein is also useful in ascreening method for identifying compounds or compositions which affectthe activity of the kinase. Thus, in another embodiment, the inventionprovides methods for identifying a composition which affects MEK6activity comprising incubating the components, which include thecomposition to be tested and the kinase or a polynucleotide encoding thekinase, under conditions sufficient to allow the components to interact,then subsequently measuring the effect the composition has on kinaseactivity or on a polynucleotide encoding the kinase. The observed effecton the kinase may be either inhibitory or stimulatory. For example, theincrease or decrease of the kinase activity can be measured by adding aradioactive compound to the mixture of components such as ³²P-ATP, andobserving radioactive incorporation into p38 or other suitablesubstrates for MEK6 to determine whether the compound inhibits orstimulates kinase activity. A polynucleotide encoding the kinase may beinserted into an expression vector and the effect of a composition ontranscription of the kinase can be measured, for example, by Northernblot analysis.

[0049] In another embodiment, the invention provides a method oftreating immunological-related cell proliferative diseases such asosteoarthritis, ischemia, reperfusion injury, trauma, certain cancersand viral disorders, and autoimmune diseases such as rheumatoidarthritis, multiple sclerosis, psoriasis, inflammatory bowel disease,and other acute phase responses. Essentially, any disorder which isetiologically linked to MEK6 kinase activity would be consideredsusceptible to treatment.

[0050] Treatment includes administration of a composition or compoundwhich modulates MEK6 kinase activity. Such modulation includes thesuppression of expression of MEK6 when it is over expressed, oraugmentation of MEK6 expression when it is under expressed. Modulationmay also include suppression of phosphorylation of p38 or relatedkinases.

[0051] As noted above, the present invention also encompassesantibodies, which may be polyclonal or monoclonal, specific for MEK6and/or one or more variants thereof Preferred antibodies are thoseantibodies that inhibit or block MEK6 activity in vivo and within a MEK6kinase assay as described above. Antibodies may be prepared by any of avariety of techniques known to those of ordinary skill in the art (see,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988). In one such technique, an immunogen comprisingthe polypeptide is initially injected into a suitable animal (e.g.,mice, rats, rabbits, sheep and goats), preferably according to apredetermined schedule incorporating one or more booster immunizations,and the animals are bled periodically. Polyclonal antibodies specificfor the polypeptide may then be purified from such antisera by, forexample, affinity chromatography using the polypeptide coupled to asuitable solid support.

[0052] Monoclonal antibodies specific for MEK6 or a variant thereof maybe prepared, for example, using the technique of Kohler and Milstein,Eur. J. Immunol. 6:511-519, 19767 and improvements thereto. Briefly,these methods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity (i.e., reactivitywith the polypeptide of interest). Such cell lines may be produced forexample, from spleen cells obtained from an animal immunized asdescribed above. The spleen cells are then immortalized by, for example,fusion with a myeloma cell fusion partner, preferably one that issyngeneic with the immunized animal. For example, the spleen cells andmyeloma cells may be combined with a nonionic detergent for a fewminutes and then plated at low density on a selective medium thatsupports the growth of hybrid cells, but not myeloma cells. A preferredselection technique uses HAT (hypoxanthine, aminopterin, thymidine)selection. After a sufficient time, usually about 1 to 2 weeks, coloniesof hybrids are observed. Single colonies are selected and tested forbinding activity against the polypeptide. Hybridomas having highreactivity and specificity are preferred.

[0053] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

[0054] Antibodies and other agents having a desired effect on MEK6activity as described above, may be administered to a patient (eitherprophylactically or for treatment of an existing disease) to modulatethe activation of p38 in vivo. For example, an agent that decreases MEK6activity in vivo may be administered to prevent or treat inflammation,autoimmune diseases, cancer or degenerative diseases. In general, foradministration to a patient, an antibody or other agent is formulated asa pharmaceutical composition which additionally comprises aphysiologically acceptable carrier. Any suitable carrier known to thoseof ordinary skill in the art may be employed in the pharmaceuticalcompositions of this invention, including the representative carriersdescribed above.

[0055] A pharmaceutical composition may also, or alternatively, containDNA encoding an antibody or other agent as described above, such thatthe active agent is generated in situ. In such pharmaceuticalcompositions, the DNA may be introduced using any of a variety ofdelivery systems known to those of ordinary skill in the art, such asthose described above. For administration of such agents, routes,frequency and doses will vary from patient to patient. In general,however, the pharmaceutical compositions may be administered asdescribed above. A suitable dose of such an agent is an amountsufficient to show benefit in the patient based on the criteria notedabove.

[0056] In a related aspect of the present invention, kits for detectingMEK6 and MEK6 kinase activity are provided. Such kits may be designedfor detecting the level of MEK6 or nucleic acid encoding MEK6, or maydetect phosphorylation of p38 in a direct kinase assay or a coupledkinase assay, in which both the level of phosphorylation and the kinaseactivity of p38 may be determined. MEK6 and MEK6 kinase activity may bedetected in any of a variety of samples, such as eukaryotic cells,bacteria, viruses, extracts prepared from such organisms and fluidsfound within living organisms. In general, the kits of the presentinvention comprise one or more containers enclosing elements, such asreagents or buffers, to be used in the assay.

[0057] A kit for detecting the level of MEK6, or nucleic acid encodingMEK6, typically contains a reagent that binds to the MEK6 protein, DNAor RNA. To detect nucleic acid encoding MEK6, the reagent may be anucleic acid probe or a PCR primer. To detect MEK6 protein, the reagentis typically an antibody. The kit also contains a reporter groupsuitable for direct or indirect detection of the reagent (i.e., thereporter group may be covalently bound to the reagent or may be bound toa second molecule, such as Protein A, Protein G, immunoglobulin orlectin, which is itself capable of binding to the reagent). Suitablereporter groups include, but are not limited to enzymes (e.g.horseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Suchreporter groups may be used to directly or indirectly detect binding ofthe reagent to a sample component using standard methods known to thoseof ordinary skill in the art.

[0058] A kit for detecting MEK6 kinase activity based on measuring thephosphorylation of p38 generally comprises p38 in combination with asuitable buffer. A kit for detecting MEK6 kinase activity based ondetecting p38 activity generally comprises p38 in combination with asuitable p38 substrate, such as ATF2. Optionally, the kit mayadditionally comprise a suitable buffer and/or material for purificationof p38 after activation and before combination with ATF2. Such kits maybe employed in direct or coupled MEK6 kinase assays, which may beperformed as described above.

[0059] In yet another aspect, MEK6 or a variant thereof may be used toidentify one or more native upstream kinases (ie., kinases thatphosphorylate and activate MEK6 in vivo). MBK6 may be used in a yeasttwo-hybrid system to identify proteins that interact with MEK6.Alternatively, an expression library may be sequenced for cDNAs thatphosphorylate MEK6.

[0060] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Cloning and Sequencing cDNA Encoding MEK6

[0061] This Example illustrates the cloning of a cDNA molecule encodingthe human MAPKK MEK6.

[0062] The Expressed Sequence Tags (EST) subdivision of the NationalCenter for Biotechnology Information (NCBI) Genbank databank wassearched with the tblastn program and the human MKK3 amino acid sequence(Derijard et al., Science 267:682685 (1995)) as query using the BLASTe-mail server. The 223 bp EST sequence F00521 displayed the highestsimilarity score. A reverse PCR primer (5′-CACATCTTCACTTGACCGAGAGCA-3′)directed against this sequence was designed with the help of the programOligo V.4.0 (National Biosciences, Inc., Plymouth, Minn.).

[0063] PolyA÷RNA was prepared from unstimulated Jurkat T cells using theMicro-Fast Track Kit (Invitrogen, San Diego, Calif.). One μg of this RNAwas used to generate an adaptor-ligated cDNA library that can be usedfor 5′ and 3′ RACE (Marathon cDNA Amplification Kit, ClontechLaboratories, Palo Alto, Calif.). The adaptor specific primer from thekit and the gene specific reverse primer were used to PCR-amplify the 5′portion of MEK6. PCR amplification was performed with a combination ofTaq and Pwo polymerases (Expand Long Template PCR System,Boehringer-Mannheim Corp., Indianapolis, Ind.) in the presence ofTaqStart antibody (Clontech Laboratories, Palo Alto, Calif.). Thismixture is designed to produce high yield of long PCR fragments andproof-reading function. All PCR amplifications were carried out in 0.2ml Perkin-Elmer thin-wall MicroAmp tubes and a Perkin-Elmer model 2400or 9600 thermocycler. The resulting 0.8 kb PCR fragnent was ligated intopGEM-T (Promega, Madison, Wis.) and sequenced (dye terminator cyclesequencing) with an ABI 373 Automated Sequencer (Applied Biosystems,Inc., Foster City, Calif.).

[0064] The sequence information from the 5′ end of the partial MEK6 cDNAwas used to design a forward PCR primer (5′-TTGTGCTCCCCTCCCCCATCAAAGGAA-3′) for 3′ RACE. The gene specific forward primer and the adaptorspecific primer were used to PCR-amplify the complete MEK6 cDNA from anadaptor-ligated MOLT-4 cDNA library. This library was generated usingone μg MOLT-4 polyA+ RNA (Clontech Laboratories, Palo Alto, Calif.) andthe Marathon cDNA Amplification Kit (Clontech Laboratories. Palo Alto,Calif.). The 1.6 kb PCR fragment was ligated into pGEM-T (Promega,Madison, Wis.) and three clones were sequenced several times on bothstrands with an ABI 373 Automated Sequencer. A BLAST search of the NCBIGenbank database for related cDNAs revealed no similar sequences. The1.6 kb cDNA encodes a potential protein of 334 amino acids with acalculated molecular weight of 37.5 kdalton.

[0065] The Bestfit program (Wisconsin Genetics Computer Group, Madison,Wis.) was used for calculating the amino acid identities between MEK6and MKK3, its closest homolog. The MacVector program (Kodak-IBI,Rochester, N.Y.) was used for aligning the amino acids of MKK3 and MEK6.MEK6 has 88% amino acid identity with MKK3, and all relevant kinasesubdomains, the ATP acceptor site and phosphorylation sites areconserved. The most divergent regions are the N-terminal region, with anadditional 18 amino acids, and the C-terminal region (FIG. 1).

Example 2 In Vivo Expression of MEK6

[0066] This Example illustrates the expression of MEK6, as compared toMKK3, in various human tissues.

[0067] Northern blots were performed using 2 μg of polyA÷RNA isolatedfrom 16 different adult human tissues, fractionated by denaturingformaldehyde 1.2% agarose gel electrophoresis, and transferred onto acharge-modified nylon membrane (Clontech Laboratories. Palo Alto,Calif.). The blots were hybridized to a MKK3 probe (700 bp MKK3 cDNAfragment) or MEK6 probe (870 bp MEK6 cDNA fragment) using ExpressHyb(Clontech Laboratories, Palo Alto, Calif.) according to themanufacturer's instructions. Both probes were prepared by labeling thecDNA with [α-³²P]dCTP (NEN, Boston, Mass.) by random priming(Stratagene, La Jolla, Calif.). For control purposes, the blots werealso hybridized to a radiolabeled β-actin probe.

[0068] The results, shown in FIGS. 2A and 2B, demonstrate that MKK3 iswidely expressed in many adult human tissues with highest levels inskeletal muscle and leukocytes (FIG. 2A). In contrast, MEK6 ispredominantly expressed in skeletal muscle and at lower levels in theheart and pancreas (FIG. 2B). No MEK6 was detected in spleen, thymus,prostate, ovary, small intestine, colon or leukocyte. All 16 tissuesanalyzed expressed equal amounts of β-actin mRNA. Some of the tissuesexpressed an MEK6-related mRNA of about 4.2 kb, which was not observedwhen MEK6 specific probe directed against the 3′ of MEK6 cDNA was used.

Example 3 Substrate Specificity of MEK6

[0069] This Example illustrates the kinase activity and substratespecificity of MEK6, as compared to MKK3, in in vitro and in vivoassays.

[0070] cDNAs encoding MEK6 and MKK3 were subcloned into a bacterialGST-fusion protein expression vector. GST-MEK6 was constructed byligating a 1.3 kb DNA fragment encoding amino acid 1 through the stopcodon of MEK6 with a serine to alanine substitution of amino acid 2 intopGEX-KG (Guan and Dixon, Ann. Biochem. 192:262-267 (1991)). Similarly,GST-MKK3, GST-p38 and GST-JNK2 were constructed by ligating therespective cDNA fragments encoding amino acid I through the stop codoninto pGEX-KG. Human p38 cDNA (Genbank accession number U10871) wascloned by PCR amplification of a Jurkat cDNA library with primersagainst the 5′ end (5′-CCAACCATGGCTCAGGAGAG-3′) and 3′ end(5′-CGGTACCTTCAGGACTCCATCT-3′) of the published human p38 sequence. Eachstrand of the PCR fragment was sequenced several times with an ABI 373Automated Sequencer. His-ERK1[K52R] was prepared as described previously(Robbins et al., J. Biol. Chem. 268:5097-5106 (1993)).

[0071] We investigated the substrate specificity of MEK6 in an in vitrokinase assay with bacterially expressed NLAPK substrates (GST-JNK2,GST-p38 and His-ERK1[K52R]). The assays were performed as previouslydescribed (Derijard et al., Cell 76:1025-1037 (1994); Lin et al.,Science 268:286-290 (1995)) with minor modifications. 0.5 μg recombinantkinase and 1 μg recombinant substrate were used, and the concentrationof [γ-³²P]ATP was 50 mM. Phosphorylated proteins were separated bySDS-PAGE on 10% gels and then subjected to autoradiography.Incorporation of [³²P]phosphate was quantitated with a phosphorimagerand Imagetuant software (Molecular Dynamics, Inc., Sunnyvale, Calif.).

[0072]FIG. 3A shows the level of autophosphorylation of the substratesGST (lane 1), GST-JNK2 (lane 2), GST-p38 (lane 3) and His-ERKCI[K52R](lane 4) and the level of phosphorylation by GST-MEK6 of the substratesGST (lane 5), GST-JNK2 (lane 6), GST-p38 (lane 7) and His-ERK1[K52R](lane 8). In each case, 1 μg of the purified recombinant substrate wasused. Autophosphorylation of MEK6 was very low compared to MKK3. MEKSautophosphorylated, whereas p38 and ERK1(K52R) did not. MEK6 efficientlyphosphor) lated p38 but none of the other substrates (FIG. 3A, comparelanes 1 to 4 with 5 to 8), although in parallel experiments thephosphorylation of JNK by JNKK has been observed (data not shown). Thisindicates that MEK6 has a substrate selectivity for the p38 subgroup ofMAPKs.

[0073] To determine whether phosphorylation of p38 is an activatingevent we analyzed the phosphorylation of recombinant ATF2 (a substratefor p38) in a coupled in vitro kinase assay. GST-ATF2 was prepared aspreviously described (Gupta et al., Science 267:389-393 (1995). FIG. 3Bshows the results of a coupled kinase assay in which purified GST orGST-MEK6 (0.1-10 μg) was incubated with purified GST-JNK2 (lanes 1 and2), GST-p38 (lanes 4 and 5) or GST (lanes 3 and 6) (0.1-10 μg) in thepresence of JNKK buffer (Lin et al., Science 268:286-290 (1995)) and 100μM ATP. The proteins were isolated by binding to GSH-sepharose and afterwashing with 20 mM HEPES (pH 7.6), 20 mM MgCl₂, incubated withGST-cJun(1-79) (lanes 1-3) or GST-ATF2 (lanes 4-6) (0.1-10 μg) in thepresence of JNK buffer with 20 M HEPES (pH 7.6), 20 mM MgCl₂, and[γ-³²P]ATP (10-500 red). Reactions were separated by SDS-PAGE andvisualized by autoradiography.

[0074] MEK6 did not cause increased phosphorylation of Jun(GST-Jun(1-79), prepared as described in Hibi et al., Genes andDevelopment 7:2135-2148 (1993)) either directly or in combination withJNK2 (FIG. 3B, lanes 1 to 3). ATF2, however, was strongly phosphorylatedby p38 that has been activated by MEK6 (FIG. 3B, lane 5). ATF2 was notdirectly phosphorylated by MEK6. These data establish that MEK6 is afunctional MAPKK in vitro and that MEK6 specifically phosphorylates p38,resulting in its activation.

[0075] Next, we examined whether MEK6 can activate p38 in vivo. Anexpression vector encoding epitope-tagged MEK6 (3xH-MEK6-SRα3) wasconstructed by replacing serine in position 2 of MEK6 with alanine,adding sequence encoding three copies of a 10 amino acid hemagglutinin(HA) epitope to the N-terminus of MEK6 and ligating the resulting cDNAinto SRα3. HeLa cells, cultured in Dulbecco's modified Eagle mediumsupplemented with 10% fetal calf serum, 500 mg/l L-glutamine, andantibiotics, were transiently transfected with 3xHA-MEK6 using calciumphosphate-mediated DNA precipitation (Graham and van der Eb, Virolog52:456-467 (1973)). Twenty-four hours later cells were stimulated withanisomycin (50 ng/mL) or UV (254 nm; 120 J/m²) for 0-120 minutes. Celllysates were prepared by solubilization in lysis buffer as described(Derijard et al., Cell 76:1025-1037 (1994)), and protein concentrationof lysates was determined by Bradford assay (Bradford, Ann. Biochem.72:248-254 (1976)).

[0076] In an initial experiment we investigated the time course of MEK6activation by anisomycin and UV treatment of transfected cells. Celllysates were used in an immune complex kinase assay with GST-p38substrate, performed as described above except that 30 μg cell lysatewas immunoprecipitated for 2 hours with the anti-HA antibody 12CA5(Boehringer-Mannheim Corp., Indianapolis, Ind.) and then incubated with1 μg of recombinant substrate. Reactions were separated by SDS-PAGE andquantitated with a phosphorimager and ImageQuant softwar. The relativelevel of MEK6 activity in untreated cells was arbitrarily assigned 1.The presence of equal amounts of MEK6 in all kinase reactions wasconfirmed by Western blot analysis (data not shown).

[0077] MEK6 activation by anisomycin as measured by its ability tophosphorylate p38, was observed as early as 10 min after treatment (FIG.4). The activation was transient and peaked at 40 min after treatment Incontrast, activation by UV was delayed by about 10 to 15 min anddeclined only slowly after a peak at 60 min (FIG. 4). Analysis of the UVdose response of MEK6 in HeLa cells revealed that doses up to 120 J/m²yielded increasing activity of MEK6 (FIG. 5).

[0078] To determine whether the increase in p38 phosphorylation byactivated MEK6 augments p38 kinase activity a coupled immune complexkinase assay was performed. Epitope-tagged MEK6 was isolated fromanisomycin-treated HeLa cells (45 minutes; 50 ng/mL) and subjected totwo subsequent kinase reactions as described above using recombinantp38, ATF2 and GST alone. In support of our in vitro results, anisomycintreatment caused increased phosphorylation of ATF2 only when MEK6 andp38 were present (FIG. 6, compare lanes 5, 6 with 7, 8). Similar resultshave been found with MEK6 activated by UV treatment of cells (data notshown). No inducible phosphorylation of p38 or ATF2 was observed in HeLacells transfected with the empty expression vector SRα3 (FIG. 6, comparelanes 5, 6 with 13, 14). This clearly indicates that the induciblephosphorylation of ATF2 depends on a kinase cascade comprised of MEK6and p38. Interestingly, p38 also phosphorylated weakly a protein with amobility slightly faster than ATF2 (indicated by * in FIG. 6). Thisphosphorylation event was slightly augmented by anisomycin in thepresence of MEK6 (FIG. 6, compare lanes 3 and 4 with lanes 11 and 12).This protein was not observed in in vitro kinase assays, and thereforeis most likely a contamination of the immunoprecipitation.

Example 4

[0079] Activation of MEH6 by Stress-Inducing Agents

[0080] This Example illustrates the response of MEK6 to a variety ofstimulators of the MAPK pathway.

[0081] To investigate the pattern of regulation of MEK6, cells weretransiently transfected with 3xHA-MEK6 (as described in Example 3) andtreated with various stimulators of the MAPK pathway. In HeLa cellsstrongest inducers of MEK6 were UV, anisomycin and NaCt followed by weakinduction with IL-1: (FIG. 7A). NGF and EGF, two strong inducers of theERK pathway, did not activate MEK6 although we noted the induciblephosphorylation of two lower molecular weight bands (see discussion).

[0082] Similar experiments were performed in COS cells, which weretransfected by the DEAE-Dextran method (Kawai and Nishizawa, Mol. Cell.Biol. 4:1172-1174 (1984)). These experiments showed a strong inductionof MEK6 by UV and to a lesser extent by anisomycin (FIG. 7B). MEK6 waspresent at equal levels in all kinase reactions as determined by WesternBlot analysis (data not shown).

[0083] These results demonstrate that MEK6 is strongly activated bystress-inducing and DNA-damaging agents, anisomycin, UV and also byosmotic shock. Phorbol esters, NGF and EGF, strong stimulators of theERK pathway did not stimulate MEK6. Similarly, cycloheximide, astimulator of p54 kinase and of the ERK pathway, did not significantlyactivate MEK6. Interestingly, we noted in our in vivo kinase assays withlysates prepared from HeLa cells, but not from COS cells, two bands ofvariable intensity that were stimulated by NGF and EGF. These bands mostlikely represent contaminants of the immunoprecipitation phosphorylatedby ERK family members.

Example 5 MEK6 is not a Physiological Substrate for MEKK

[0084] This Example evaluates the ability of MEKK to phosphorylate MEK6as compared to its ability to phosphorylate JNKK.

[0085] MEKK has been described as a MAPKKK leading to thephosphorylation and activation of JNKK (Lin et al., Science 268:286-290(1995); Minden et al., Science 266:1719-1722 (1994); Yan et al., Nature372:798-800 (1994)). In an initial experiment, HeLa (FIG. 7A) or COS(FIG. 7B) cells were transiently transfected with epitope-tagged MEK6(lanes 1 to 12) or the empty expression vector SRα3 (lanes 13 to 16) andtreated for 45 min with IL-1p (10 ng/ml), TNF-α (10 ng/ml), EGF (50ng/ml), NGF (50 ng/ml), PMA (50 ngZ/ml), Anisomycin (50 ng/ml),Cycloheximide (CX, 50 ng/ml), Arsenite (200 μM), NaCl (200 μM), UV (254nm; 120 J/m²) or cotransfected with 1000 ng CMV5-MEKK as indicated. Celllysates were used in an immune complex kinase assay with GST-p38substrate as described in Example 3. MEK6 activity was quantitated witha phosphorimager and ImageQuant software. The presence of equal amountsof MEK6 in all kinase reactions was confused by Western blot analysis(data not shown).

[0086] With 1000 ng cotransfected expression vector for MEKK, weobserved stimulation of MEK6 activity in COS cells but not HeLa cells(FIG. 7A, lane 12, FIG. 7B, lane 12). This prompted us to examine morecarefully whether MEKK is able to phosphorylate MEK6. COS cells weretransiently transfected with increasing amounts of expression vectorencoding MEKK in the presence of a constant amount of expression vectorencoding epitope-tagged MEK6 (FIG. 8, lanes 1 to 7) or JNKK (FIG. 8,lanes 8 to 12), and increasing amounts of CMV5-MEKK expression vector asindicated in FIG. 8. Cell lysates were used in an immune complex kinaseassay with GST-p38 (lanes 1 to 7) or GST-JNK2 (lanes 8 to 12) substrateas described in Example 3. Kinase activity was quantitated with aphosphorimager and ImageQuant software.

[0087] We observed strong JNKK activation in cells transfected with aslittle as 125 ng of the MEKK expression vector. Comparable amounts ofMEK6 activation, however, were not observed until 1000 ng of the MEKKexpression vector were cotransfected. These data suggest that MEKK doesnot participate in the kinase cascade consisting of MEK6 and p38.

[0088] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

1 6 1 1002 DNA homo sapien CDS (1)...(1002) 1 atg tct cag tcg aaa ggcaag aag cga aac cct ggc ctt aaa att cca 48 Met Ser Gln Ser Lys Gly LysLys Arg Asn Pro Gly Leu Lys Ile Pro 1 5 10 15 aaa gaa gca ttt gaa caacct cag acc agt tcc aca cca cct cga gat 96 Lys Glu Ala Phe Glu Gln ProGln Thr Ser Ser Thr Pro Pro Arg Asp 20 25 30 tta gac tcc aag gct tgc atttct att gga aat cag aac ttt gag gtg 144 Leu Asp Ser Lys Ala Cys Ile SerIle Gly Asn Gln Asn Phe Glu Val 35 40 45 aag gca gat gac ctg gag cct ataatg gaa ctg gga cga ggt gcg tac 192 Lys Ala Asp Asp Leu Glu Pro Ile MetGlu Leu Gly Arg Gly Ala Tyr 50 55 60 ggg gtg gtg gag aag atg cgg cac gtgccc agc ggg cag atc atg gca 240 Gly Val Val Glu Lys Met Arg His Val ProSer Gly Gln Ile Met Ala 65 70 75 80 gtg aag cgg atc cga gcc aca gta aatagc cag gaa cag aaa cgg cta 288 Val Lys Arg Ile Arg Ala Thr Val Asn SerGln Glu Gln Lys Arg Leu 85 90 95 ctg atg gat ttg gat att tcc atg agg acggtg gac tgt cca ttc act 336 Leu Met Asp Leu Asp Ile Ser Met Arg Thr ValAsp Cys Pro Phe Thr 100 105 110 gtc acc ttt tat ggc gca ctg ttt cgg gagggt gat gtg tgg atc tgc 384 Val Thr Phe Tyr Gly Ala Leu Phe Arg Glu GlyAsp Val Trp Ile Cys 115 120 125 atg gag ctc atg gat aca tca cta gat aaattc tac aaa caa gtt att 432 Met Glu Leu Met Asp Thr Ser Leu Asp Lys PheTyr Lys Gln Val Ile 130 135 140 gat aaa ggc cag aca att cca gag gac atctta ggg aaa ata gca gtt 480 Asp Lys Gly Gln Thr Ile Pro Glu Asp Ile LeuGly Lys Ile Ala Val 145 150 155 160 tct att gta aaa gca tta gaa cat ttacat agt aag ctg tct gtc att 528 Ser Ile Val Lys Ala Leu Glu His Leu HisSer Lys Leu Ser Val Ile 165 170 175 cac aga gac gtc aag cct tct aat gtactc atc aat gct ctc ggt caa 576 His Arg Asp Val Lys Pro Ser Asn Val LeuIle Asn Ala Leu Gly Gln 180 185 190 gtg aag atg tgc gat ttt gga atc agtggc tac ttg gtg gac tct gtt 624 Val Lys Met Cys Asp Phe Gly Ile Ser GlyTyr Leu Val Asp Ser Val 195 200 205 gct aaa aca att gat gca ggt tgc aaacca tac atg gcc cct gaa aga 672 Ala Lys Thr Ile Asp Ala Gly Cys Lys ProTyr Met Ala Pro Glu Arg 210 215 220 ata aac cca gag ctc aac cag aag ggatac agt gtg aag tct gac att 720 Ile Asn Pro Glu Leu Asn Gln Lys Gly TyrSer Val Lys Ser Asp Ile 225 230 235 240 tgg agt ctg ggc atc acg atg attgag ttg gcc atc ctt cga ttt ccc 768 Trp Ser Leu Gly Ile Thr Met Ile GluLeu Ala Ile Leu Arg Phe Pro 245 250 255 tat gat tca tgg gga act cca tttcag cag ctc aaa cag gtg gta gag 816 Tyr Asp Ser Trp Gly Thr Pro Phe GlnGln Leu Lys Gln Val Val Glu 260 265 270 gag cca tcg cca caa ctc cca gcagac aag ttc tct gca gag ttt gtt 864 Glu Pro Ser Pro Gln Leu Pro Ala AspLys Phe Ser Ala Glu Phe Val 275 280 285 gac ttt acc tca cag tgc tta aagaag aat tcc aaa gaa cgg cct aca 912 Asp Phe Thr Ser Gln Cys Leu Lys LysAsn Ser Lys Glu Arg Pro Thr 290 295 300 tac cca gag cta atg caa cat ccattt ttc acc cta cat gaa tcc aaa 960 Tyr Pro Glu Leu Met Gln His Pro PhePhe Thr Leu His Glu Ser Lys 305 310 315 320 gga aca gat gtg gca tct tttgta aaa ctg att ctt gga gac 1002 Gly Thr Asp Val Ala Ser Phe Val Lys LeuIle Leu Gly Asp 325 330 2 333 PRT homo sapien 2 Ser Gln Ser Lys Gly LysLys Arg Asn Pro Gly Leu Lys Ile Pro Lys 1 5 10 15 Glu Ala Phe Glu GlnPro Gln Thr Ser Ser Thr Pro Pro Arg Asp Leu 20 25 30 Asp Ser Lys Ala CysIle Ser Ile Gly Asn Gln Asn Phe Glu Val Lys 35 40 45 Ala Asp Asp Leu GluPro Ile Met Glu Leu Gly Arg Gly Ala Tyr Gly 50 55 60 Val Val Glu Lys MetArg His Val Pro Ser Gly Gln Ile Met Ala Val 65 70 75 80 Lys Arg Ile ArgAla Thr Val Asn Ser Gln Glu Gln Lys Arg Leu Leu 85 90 95 Met Asp Leu AspIle Ser Met Arg Thr Val Asp Cys Pro Phe Thr Val 100 105 110 Thr Phe TyrGly Ala Leu Phe Arg Glu Gly Asp Val Trp Ile Cys Met 115 120 125 Glu LeuMet Asp Thr Ser Leu Asp Lys Phe Tyr Lys Gln Val Ile Asp 130 135 140 LysGly Gln Thr Ile Pro Glu Asp Ile Leu Gly Lys Ile Ala Val Ser 145 150 155160 Ile Val Lys Ala Leu Glu His Leu His Ser Lys Leu Ser Val Ile His 165170 175 Arg Asp Val Lys Pro Ser Asn Val Leu Ile Asn Ala Leu Gly Gln Val180 185 190 Lys Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser ValAla 195 200 205 Lys Thr Ile Asp Ala Gly Cys Lys Pro Tyr Met Ala Pro GluArg Ile 210 215 220 Asn Pro Glu Leu Asn Gln Lys Gly Tyr Ser Val Lys SerAsp Ile Trp 225 230 235 240 Ser Leu Gly Ile Thr Met Ile Glu Leu Ala IleLeu Arg Phe Pro Tyr 245 250 255 Asp Ser Trp Gly Thr Pro Phe Gln Gln LeuLys Gln Val Val Glu Glu 260 265 270 Pro Ser Pro Gln Leu Pro Ala Asp LysPhe Ser Ala Glu Phe Val Asp 275 280 285 Phe Thr Ser Gln Cys Leu Lys LysAsn Ser Lys Glu Arg Pro Thr Tyr 290 295 300 Pro Glu Leu Met Gln His ProPhe Phe Thr Leu His Glu Ser Lys Gly 305 310 315 320 Thr Asp Val Ala SerPhe Val Lys Leu Ile Leu Gly Asp 325 330 3 27 DNA Artificial Sequence 5′specific forward primer 3 ttgtgctccc ctcccccatc aaaggaa 27 4 24 DNAArtificial Sequence Reverse 5′ PCR primer 4 cacatcttca cttgaccgag agca24 5 20 DNA Artificial Sequence 5′ primers of human p38 5 ccaaccatggctcaggagag 20 6 22 DNA Artificial Sequence 3′ Primer of human p38 6cggtaccttc aggactccat ct 22

1. A polypeptide comprising the amino acid sequence provided in SEQ IDNO:2 or a variant thereof that differs only in conservativesubstitutions and/or modifications at no more than 10% of the amino acidresidues.
 2. A constitutively active variant of a polypeptide accordingto claim
 1. 3. A polypeptide comprising the amino acid sequence providedin SEQ ID NO:2 modified at no more than 10% of the amino acid residues,such that said polypeptide is rendered constitutively inactive.
 4. Anisolated DNA molecule encoding a polypeptide according to any of claims1-3.
 5. An isolated DNA molecule comprising the nucleotide sequenceprovided in SEQ ID NO:1.
 6. A recombinant expression vector comprising aDNA molecule according to claim
 4. 7. A host cell transformed ortransfected with an expression vector according to claim
 6. 8. A methodfor phosphorylating p38 comprising contacting p38 with a polypeptideaccording to either of claims 1 or 2, thereby phosphorylating p38.
 9. Amethod for activating a member of the p38 cascade in an organism,comprising administering to an organism a polypeptide according toeither of claims 1 or 2, thereby activating a member of the p38 cascade.10. The method of claim 9 wherein the member of the p38 cascade is p38.11. A method for screening for an agent that inhibits signaltransduction via the p38 cascade, comprising: (a) contacting a candidateagent with a polypeptide according to either of claims 1 or 2; and (b)subsequently measuring the ability of said polypeptide to activate p38,and thereby evaluating the ability of the compound to inhibit signaltransduction via the p38 cascade.
 12. A method for screening for anagent that stimulates signal transduction via the p38 cascade,comprising: (a) contacting a candidate agent with a polypeptideaccording to either of claims 1 or 2; and (b) subsequently measuring theability of said polypeptide to activate p38, and thereby evaluating theability of the compound to stimulate signal transduction via the p38cascade.
 13. A monoclonal antibody that binds to a polypeptide accordingto either of claims 1 or
 2. 14. A monoclonal antibody according to claim13, wherein said antibody inhibits the phosphorylation of p38 by saidpolypeptide.
 15. A method for treating a patient afflicted with adisease associated with the p38 cascade, comprising administering to apatient a compound that inhibits the phosphorylation of p38 by MEK6. 16.The method of claim 15 wherein said compound is a monoclonal antibody.17. The method of claim 15 wherein said compound comprises a nucleotidesequence.
 18. A method for detecting MEK6 kinase activity in a samplecomprising evaluating the ability of the sample to phosphorylate p38,thereby detecting MEK6 kinase activity in the sample.
 19. A kit fordetecting MEK6 kinase activity in a sample, comprising p39 incombination with MEK6 and a suitable buffer.
 20. A method foridentifying a composition which affects MBK6 kinase activity,comprising: (a) incubating the composition and MEK6 kinase orpolynucleotide encoding the kinase, wherein the step of incubation iscarried out under conditions and for a time sufficient to allow thecomponents to interact; and (b) measuring the effect of the compositionon MEK6 kinase or polynucleotide encoding the kinase.
 21. A method oftreating an immunologically related disorder associated with MEK6 kinaseactivity, comprising administering to a subject having the disorder atherapeutically effective amount of a compound which modulates MEK6kinase activity.